Functional and morphological changes observed on CCCP-treated platelets resemble platelet storage lesion. (A) Washed mouse platelets were treated with DMSO (control) or CCCP for 60 minutes. Platelets were immediately fixed, permeabilized, and stained for tubulin-β1 and tubulin-β2, and tubulin-α. DIC indicates differential interference contrast images; tubulin stain, immunofluorescence images. Original magnification, × 1000. (B,C) Platelets were stained with annexin V-FITC or RB40.34-FITC (anti-P-selectin) and immediately analyzed on a FACScalibur. 0 indicates DMSO; 30,60,90, platelets treated with CCCP for 30, 60, or 90 minutes; A23, platelets activated for 10 minutes with 50 μg/mL A23187; thr, platelets activated for 10 minutes with 0.5 U/mL thrombin. (D) Dual-color analysis of control (DMSO) and CCCP-treated platelets stained for GPIbα and GPIbβ. (E) Immunoblot analysis of platelet lysates. Cells were treated for 60 minutes with DMSO (control) or CCCP, lysed by addition of 2 × SDS sample buffer, and proteins were separated by 10% SDS-PAGE. Results are representative of 5 individual experiments.